体外分化胚胎干细胞为造血干细胞重建造血功能的研究

In vitro hematopoietic stem cells from embryonic stem cells reconstruct hematopoiesis in mice

目的:探讨体外定向分化胚胎干细胞(ESCs)为造血干细胞(HSCs)对体内造血功能的重建作用。方法:将小鼠E14.1胚胎干细胞采用"三步诱导法"在体外分化发育为HSCs,造血克隆形成(CFU)实验观察其体外造血集落形成情况,免疫磁珠分选纯化HSCs移植给经亚致死剂量γ射线照射的雌性SCID小鼠,观察其植入及小鼠造血功能恢复情况。结果:经过分阶段诱导,多种造血刺激因子联合应用能有效促进ESCs定向分化发育为HSCs,流式细胞仪检测HSCs特异性表面标志物CD34+/Sca-1+表达最高为(58.64±4.20)%,CFU培养能形成较多的红系、粒系/巨噬细胞系及混合细胞集落,Wright-Giemsa染色显示为原始的造血细胞。此阶段的HSCs经分选纯化后移植给经γ射线照射后的小鼠,移植组小鼠+10d造血功能开始恢复,观察40d后除血小板恢复较慢外,白细胞、红细胞、血红蛋白等指标已接近正常,植入率为71.4%,存活率为43.0%,染色体检测证实已由受体鼠的XX转为供体鼠的XY。结论:采用分阶段诱导的方法,可在体外定向诱导小鼠ESCs分化发育为HSCs,此来源的HSCs可以有效重建体内造血功能。

AIM： To investigate the potential of hematopoietic stem cells （HSCs） derived from mouse embryonic stem cells （ESCs） to reconstruct hematopoiesis in vivo. METHODS： Using a three -step method, a mice embryonic stem cell line, E14.1 was induced into hematopoietic stem cells. The cell markers with CD34＋/Sca - 1＋ were identified by flow cytometry analysis, then HSCs （ 1 × 10^9 cells/L） from third - step were injected into SCID mice for observing teratoma formation. To validate function of HSCs, colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted. RESULTS ： The method of three - step differentiation, combined to use more hematopoietic stimulating factor promoted the El4. 1 cell differentiation into HSCs with highest percent of CD34＋/Sca - 1＋ cells （ as high as 58.64% ± 4. 20% ） with more CFU - E, CFU - GM and CFU - GEMM populations. The cells showed the character of hematopoietic progenitors by Wright - Giemsa staining. Positive selected CD34 ＋/Sca - 1 ＋ cells by magnetic sorting from third - step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation, with 71.4% （5/7） successful engraftment rate. Three recipients showed that the cell population of the peripheral blood leukocytes, red blood cells and hemoglobin approached to normal index at 40 d after transplantation, but followed relative slow renew in platelet count. Survival rate in transplant group was 43%, compared to 100% mortality in control mice. Karyotyping assays confirmed the female mice with XY. CONCLUSION： The three - step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs. HSCs derived from mouse ESCs can reconstruct hematopoiesis.